Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.
Atherosclerosis and angiogenesis
A vessel wall is composed of an endothelial cells monolayer (intima), lining the lumen of the artery and directly in contact with the circulating blood cells. Under this monolayer, smooth muscle cells (media) and fibroblasts (adventitia) constitute the mural structure of the artery.
Atherosclerosis is an inflammatory disease and one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque due to high plasma concentrations of cholesterol, in particular those of LDL (low-density lipoprotein), the accumulation of lipids, waste of necrotic cells and a series of specific cellular and molecular responses within artery wall . The adventitia and the outer layers of media of an atherosclerosis-prone arterial wall are vascularized by vasa vasorum .
The lesions of atherosclerosis mostly occur in large or medium-sized elastic and muscular arteries. Many studies have demonstrated that atherosclerosis is affected by local angiogenesis. A decrease in blood flow causes a reduction in nutrient intake and oxygen that can lead to ischaemia of heart, brain or other organs, resulting in tissue damage .
In pathological atherosclerosis, the endothelial cells of vessels are the first to be injured. During the development of this disease, extracellular matrix digestion by proteases facilitates the migration of endothelial cells, smooth muscle cells and fibroblasts, but also results in the release of growth factors such as FGF (fibroblast growth factor) and VEGF (vascular endothelial growth factor), which otherwise would remain sequestered within the matrix. Among proteases, over 20 MMPs (matrix metalloproteinases) have been described and implicated in angiogenesis and cell proliferation in atherosclerosis . Vascular remodelling occurring during both angiogenesis and atherosclerosis are orchestrated by VEGF, which directly increases the secretion of MMPs, urokinase-type and tissue-type plasminogen activators (uPA and tPA respectively), and PAI-1 (plasminogen-activator inhibitor 1) by endothelial cells. Together, these proteases and their inhibitors create a balance that is necessary for proper cleavage of the insoluble fibrin clot and the migration of infiltrating cells during the formation of the atherosclerotic plaque [5,6]. Moreover, upon growth of an atherosclerotic lesion in the intima, neovascular sprouts originating from the adventitial vasa vasorum enter the lesion, the local pro-angiogenic microenvironment in the lesion being created by intramural hypoxia, increased intramural oxidant stress and inflammatory cell infiltration (macrophages, T-cells and mast cells). The angiogenic factors that are present in the lesions include various growth factors, chemokines, cytokines, proteases and several other factors possessing direct or indirect angiogenic activities, whereas the current list of anti-angiogenic factors is smaller . An imbalance between endogenous stimulators and inhibitors of angiogenesis, with a predominance of the inhibitory ones, is essential for the development of neovessels during the progression of the lesion. Neovascularization provides oxygen and nutrients to the cells of atherosclerotic lesions, therefore initially preventing cell death and contributing to plaque growth and stabilization. Nevertheless, inflammatory cells may induce rupture of the fragile neovessels, and then cause intraplaque haemorrhage and destabilize the atherosclerotic plaque [7,8].
Angiogenesis is a process characterized by a combination of sprouting of new vessels from the sides and ends of pre-existing ones or by longitudinal division of existing vessels with peri-endothelial cells obtained by intussusceptions, which may then split and branch into pre-capillary arterioles and capillaries. Depending on the ultimate fate with respect to the type of vessel (artery or vein, capillary), activated endothelial cells can migrate and proliferate to form new vessels, forming anastomic connections with each other .
The blood vessels and heart are the first organs to form and function during mammalian development, but the vasculature continues to remodel dynamically even as the organs function. Many of the events that occur during vascular development in the embryo are recapitulated during neovascularization in the adult such as that in the wound healing and repair process. In pathological states such as tumorigenesis, host blood vessels are recruited to grow into the vicinity of the tumour to maintain its growth and promote potential routes for metastasis .
It was demonstrated that inhibition of expression of a pro-angiogenic factor, VEGF, decreased the progression of atherosclerosis . Other studies indicate that bone-marrow-derived endothelial cells can be mobilized to peripheral blood by a therapeutic dose of angiogenic growth factors such as VEGF and PDGF (platelet-derived growth factor) . These factors, originally recognized to promote both angiogenesis and vasculogenesis in the embryo, might also promote mobilization of EPCs (endothelial precursor cells) from bone marrow in adults . Although VEGF has been considered relatively specific for endothelial cells , it also probably affects monocyte activation and migration via a primary effect on the receptor Flt-1 (Fms-like tyrosine kinase-1)/VEGFR1 (VEGF receptor 1) in bone marrow and peripheral blood [15,16].
Many other growth factors such as FGF, PDGF and Ang1 (angiopoietin-1) regulate angiogenesis , and chemokines have been shown recently to play a role in this process. Growth factors and chemokines induce the recruitment of macrophages, platelets and leucocytes on endothelial cell surface, and therefore increase the inflammation and favour the formation of atherosclerotic plaque. Vasa vasorum neovascularization favours the entry of pro-inflammatory components such as chemokines into the coronary vessel wall, leading to the recruitment of progenitor cells from the bone marrow to form neovessels.
Chemokines are a large family of small secreted proteins that are classified depending on the spacing or presence of four conserved cysteine residues: CC, CXC, CX3C and C chemokines . The first chemokines discovered were IL-8 (interleukin-8)/CXCL (CXC chemokine ligand) 8, PF4 (platelet factor 4)/CXCL4 and MCP-1 (monocyte chemoattractant protein 1)/CCL (CC chemokine ligand) 2, which were isolated in the 1980s as factors secreted during inflammation, and it was recognized that they attracted specific phagocytes and monocytes . Chemokines have many physiological roles; they are implicated in haemopoiesis , tissue repair by the ability to induce proliferation and cell migration  and embryonic development by the ability to stimulate the formation of new vessels . In addition to their structural classification, chemokines have been classified depending on their function, as inflammatory or homoeostatic chemokines .
In atherosclerosis, leucocyte recruitment induced by inflammatory chemokines is mediated via their high- and low-affinity interactions with GPCR (G-protein-coupled receptor) and GAG (glycosaminoglycan) chains respectively that are present on the endothelial cell surface. These ligand–receptor interactions induce firm adhesion and trigger transendothelial migration [19,26]. GAG chains are either present in the extracellular matrix or displayed by HSPGs (heparan sulfate proteoglycans) present at the cell surface. The collateral flow induced the expression of adhesion molecules [ICAM-1 (intercellular adhesion molecule 1)] and the expression of chemotactic molecules such as MCP-1/CCL2 by endothelial cells involved in monocyte adherence .
It has been established that inflammatory cells are indirectly implicated in the growth of vasa vasorum, after the occlusion of the artery in the myocardium . Indeed, inflammatory cells activate endothelial cells inducing the up-regulation of basic FGF, PDGF and TGFβ (transforming growth factor β), which stimulate smooth muscle cell growth and vasa vasorum enlargement to develop the atherosclerotic plaque. Moreover, studies demonstrate that monocyte expression of the MCP-1/CCL2 receptor CCR (CC chemokine receptor) 2 is stimulated by hypercholesterolaemia and monocytes derived from hypercholesterolaemic patients exhibit increased chemotactic responses to MCP-1/CCL2 .
Chemokines and angiogenesis
Some chemokines have been shown to contribute to angiogenesis by stimulating endothelial cell branching and the formation of numerous long capillary sprouts in angiogenesis assays. The main chemokines studied in angiogenesis are CXC and CC chemokines.
The CXC chemokines can be further divided into two groups of molecules (ELR+ and ELR−) according to the presence or absence of an ELR (Glu-Leu-Arg) motif located immediately before the first cysteine residue. Several members of CXC family are among the first chemokines identified as regulators of angiogenesis, acting either as angiogenic or angiostatic factors. Interestingly, the presence or the absence of an ELR motif seems to correlate with an angiogenic activity; indeed, chemokines with ELR motif exhibit pro-angiogenic properties, whereas anti-angiogenic properties are associated with the ELR− chemokines, except the SDF-1 (stromal-cell-derived factor 1)/CXCL12 chemokine .
To consolidate this, it has been shown that the interaction of SDF-1/CXCL12 with the GPCR CXCR4 (CXC chemokine receptor 4) on endothelial cells amplifies angiogenesis further by recruiting endothelial progenitor cells during wound healing of peripheral limbs after an ischaemia .
Some studies suggest that the chemokines SDF-1/CXCL12, MCP-1/CCL2 and RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 would regulate angiogenesis . Indeed, in in vivo models, SDF-1/CXCL12 would recruit EPCs to murine ischaemic leg . In an in vitro model, the incubation of endothelial cells from different organs with the chemokines SDF-1/CXCL12, RANTES/CCL5 and lymphoid tissue chemokine [SLC (secondary lymphoid tissue chemokine)/CXCL21] results in a rearrangement of these cells and the formation of pseudo-vessels of human appendix endothelial cells .
Angiogenic properties of RANTES/CCL5
RANTES/CCL5 is a low-molecular-mass (7.8 kDa) chemokine, which is involved in chronic inflammation by the recruitment of inflammatory cells [25,33]. RANTES/CCL5 is secreted by many cell types such as endothelial cells, smooth muscle cells, macrophages, platelet and activated T-cells. RANTES/CCL5 binds to chemokine receptors (GPCRs) CCR1, CCR3 and CCR5 [33,34].
RANTES/CCL5 expression is associated with, and may play a role in, chronic inflammation , wound repair  and the progression of some angiogenesis-dependent tumours , by the ability to induce leucocyte recruitment and to activate these cells . These effects of RANTES/CCL5 are consistent with a role for the chemokine on the process of inflammatory angiogenesis. In the atherosclerosis disease, RANTES/CCL5 is released mainly by platelet and smooth muscle cells. By its ability to bind to CCR1 and CCR5 GPCRs expressed on T-cells or monocytes, RANTES/CCL5 may lead to the adhesion and the transmigration of T-cells and monocytes through the endothelial wall [34,39].
In addition, in a model of sponge-induced inflammatory angiogenesis, a dynamic expression of the chemokine RANTES/CCL5 was found to be correlated with neovascularization and inflammatory cell accumulation . Surprisingly, exogenous RANTES/CCL5 in the early stage of the process actually prevented angiogenesis. CCR1 and CCR5 are the main receptors of RANTES/CCL5. Thus a deletion of CCR5 alone will still allow RANTES/CCL5 to bind to CCR1 . However, exogenous RANTES/CCL5 no longer modified angiogenesis in Ccr5−/− mice, showing that RANTES/CCL5 signals mainly through CCR5 in this model. Thus the activation of CCR5 by exogenous RANTES/CCL5 may initiate a cascade of events leading to inhibition of sponge-induced angiogenesis .
Controversially, in a recent study, Braunersreuther et al.  showed that the injection of a RANTES/CCL5 antagonist, [A44ANA47]-RANTES/CCL5, reduced the infarct size in an in vivo model of ischaemia and reperfusion in Apoe−/− mice after ligature of the left coronary artery. This beneficial effect of [A44ANA47]-RANTES/CCL5 treatment was associated with reduced leucocyte infiltration into the reperfused myocardium, as well as decreased CC chemokines MCP-1/CCL2 and MIP-1α/CCL3 expression, oxidative stress and apoptosis . Moreover, mice that were deficient for the RANTES/CCL5 receptor CCR5 did not exhibit myocardium salvage in a model of ischaemia/reperfusion . Furthermore, [A44ANA47]-RANTES/CCL5 did not mediate cardioprotection in these Apoe−/−/Ccr5−/− mice, probably due to an enhanced expression of compensatory chemokines. Thus RANTES/CCL5 inhibition may exert cardioprotective effects during early myocardial reperfusion, through its anti-inflammatory properties. Therefore blocking chemokine receptor–ligand interactions might become a novel therapeutic strategy to reduce reperfusion injuries in patients during acute coronary syndromes. The use of an another antagonist, Met-RANTES/CCL5, impaired for GPCR binding, reduces the homing of endothelial progenitor cells, a step that is essential to endothelium building and the induction of neo-angiogenesis cells .
These results are supported by a study on RANTES/CCL5 delivery using a disc system in a mouse model to induce angiogenesis and by the interaction on both RANTES/CCL5 receptors CCR1 and CCR5 which are implicated in the recruitment of neutrophils within post-ischaemic tissue in an animal model .
Atherosclerosis is a complex inflammatory disease. The progression of atherosclerotic plaques involves many different factors. In the last step, angiogenesis destabilizes and contributes to the plaque rupture. The complex array of anti-angiogenic and pro-angiogenic factors which interact with multiple cells and tissues has to be tightly regulated. Therapeutic strategies may target anti-angiogenic factors in order to prevent tumorigenesis or pro-angiogenic factors, thus preventing ischaemic diseases .
Chemokines may exert their regulatory activity on angiogenesis directly or as a consequence of leucocyte infiltration and/or the induction of growth factor expression . The chemokine–receptor interaction is crucial for both arrest and transmigration of inflammatory cells through the endothelium . Binding of RANTES/CCL5 to GAGs and to GPCRs are crucial for its pro-inflammatory activity [42–44]. Mutating the main GAG-binding domain in RANTES has been shown to switch the [A44ANA47]-RANTES/CCL5 chemokine to a potent anti-inflammatory molecule in murine models of inflammatory diseases . The anti-inflammatory properties of a RANTES/CCL5 mutant may be associated with a cardioprotective effect of RANTES/CCL5 inhibition during early myocardial reperfusion. These results indicate that blocking chemokine receptor–ligand interactions might become a novel therapeutic strategy to reduce reperfusion injuries in patients during acute coronary syndromes.
The present review indicates the involvement of chemokines in angiogenesis, notably RANTES/CCL5, mostly known for its inflammatory properties. The pro- or anti-angiogenic effect of RANTES/CCL5 is still controversial. It has been demonstrated that RANTES/CCL5 displayed an anti-angiogenic activity in a sponge-induced angiogenesis model . In contrast, several studies suggest that RANTES/CCL5 may be considered as a pro-angiogenic factor. Inflammatory cells recruited by the binding of RANTES/CCL5 on CCR5 may secrete growth factors involved in vessel formation or in endothelial progenitor cell recruitment [42,45]. Chemokines may induce the migration and the proliferation of endothelial cells , thus promoting the angiogenesis. In a mouse model of surgically induced hind limb ischaemia, RANTES/CCL5 exerts a pro-angiogenic effect .
Finally, innovative gene technologies as miRNA (microRNA) and advances in animal modelling may be useful to make a major advance in our understanding of the relevance of chemokines in angiogenesis.
Advances in the Cellular and Molecular Biology of Angiogenesis: A Biochemical Society/Wellcome Trust Focused Meeting held at the University of Birmingham, U.K., 27–29 June 2011. Organized and Edited by Stuart Egginton and Roy Bicknell (Birmingham, U.K.).
CC chemokine ligand
CC chemokine receptor
CXC chemokine ligand
endothelial precursor cell
fibroblast growth factor
monocyte chemoattractant protein 1
platelet-derived growth factor
regulated upon activation, normal T-cell expressed and secreted
stromal-cell-derived factor 1
vascular endothelial growth factor